Linear molecular weight calibration curve

Those which employ one or more broad MWD standards with known M or [n] and assume a linear molecular weight calibration curve and finally those which employ one broad MWD... 

Methods which assume a linear molecular weight calibration curve will now be briefly considered. 

Methods Based on a Linear Molecular Weight Calibration Curve... 

The assumed form of the linear molecular weight calibration curve is given in equation (lO)... 

We now move on to aqueous SEC and the use of the method of calibration based on a linear molecular weight calibration curve and two broad MWD polydext ran s with known and M. ... 

Linear type columns are especially designed to have wider linear molecular weight ranges. These linear-type columns are highly recommended for correcting nonlinear sections of molecular weight calibration curves (Table 6.2). 

The second method assumes that the molecular weight calibration curve is linear on a semilog plot and should be employed where universal calibration is not practical as with aqueous SEC. Several variants of these methods involving different molecular weight data for the standards are discussed. The proposed methods have been evaluated using aqueous SEC and polydextran standards and nonaqueous SEC with polyvinylchloride standards. 

Figure 4. Molecular weight calibration curves for polydextran obtaimd u g linear calibration and two broad M WD standards and by plotting Mrm ( / Mif Mw) vs, peak retention volume ((%) Mrms, (---------) M(v) = 0,37 X exp(—OJ v))...

Peedc-to-peak resolution in SEC can be calculated by the ratio of peak separation at the peak maxiaut to the sum of the baseline peak widths. This general definition of resolution is less useful in SEC, where a measure of the ability of the column to separate solutes of different molecular weight is required. For this purposes, we define a new term, the specific resolution factor, R, which relates peak resolution to sample molecular weight, assuming all measurements are made within the linear region of the molecular weight calibration curve, equation (4.41)... 

Non-Aqueous SEC Experiments. Non-aqueous SEC separations were carried out at ambient temperatures using two Varian MicroPak TSK GMH6 columns in series (7.5mm i.d. x 30cm each). This column is a mixed bed column containing pore sizes from 250 8 to 107 blended to ensure linearity of the molecular weight calibration curve. The mobile phase employed tetrahydrofuran at a flow rate of 1 ml/mln. Sample injection volumes were 50p)l using a Rheodyne 7126 manual loop Injector. 

Aqueous SEC Experiments. Aqueous SEC separations were carried out at ambient temperature using two column sets of MicroPak TSK PW Type gel which were investigated for linearity of molecular weight calibration curve using polyethylene glycol (PEG) and polyethylene oxide (PEO) narrow MWD standards. Columns were matched in pore volume as closely as possible to promote linearity of the molecular weight calibration cuiA e. Column set A consisted... 

Universal calibration methodology has been successfully applied to PVC and many other chemical types of polymers (22). However, availability of Mark-Houwlnk constants can limit the utility of universal calibration. In these cases, the use of a linear, polydlsperse standard calibration method Is a viable alternative for generation of a molecular weight calibration curve. 

The linear calibration method provides an equivalent molecular weight calibration curve to a peak position method of calibration using a series of polystyrene standards. 

The specific resolution factor, Rsp in SEC, relates peak separations to sample molecular weight, assuming all measurements are made within the linear region of the molecular weight calibration curve, Eq. (4.30)... 

Typically, the elution of small molecules has been described as a linear function of log M and the elution volume, Vg. Molecular weight calibration curves obtained with members of a specific homologous series often give very good linearity with remaining members of the series, within the limit of the included volume of the column system used. An example of this application with oligosaccharides and several monosaccharides is shown in Figure 1. The predictive ability of such log M curves decreases as the solute studied... 

FIGURE 22.10 (a) Immunoblot using polyclonal anti-lupin antibody (1, molecular-weight marker 2, L. aftMj-derived extract 3, L. angustifolius flour extract) (b) Representative linear six-point calibration curve based on the standard curve obtained using the lupin protein standard in the sandwich ELISA. (From Holden etal., 2005.) (Reprinted with permission from Reference Citation. Copyright 2005 American Chemical Society.)... 

In summary, the various non-size exclusion effects operating In SEC of proteins affect the linearity of the calibration curve unfavourably and make an assessment of reliable molecular weight data difficult. In the most favourable case, a standard deviation of 15% In the log-linear calibration plot for proteins was reported on a TSK gel 3000 SW column. ... 

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